Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C
| Autor: | Werner Aretz, Klaus Sauber, Wolfgang Kabsch, Karin Fritz-Wolf, Alexander Liesum, Gudrun Lange, Herman Schreuder, Klaus Peter Koller |
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| Jazyk: | angličtina |
| Rok vydání: | 2002 |
| Předmět: |
Models
Molecular Stereochemistry Molecular Sequence Data Penicillin amidase Crystallography X-Ray Biochemistry Article Catalysis Amidohydrolases Serine chemistry.chemical_compound Catalytic triad Hydrolase Amino Acid Sequence Molecular Biology Histidine Cephalosporin Antibiotic chemistry.chemical_classification Cephalosporin C Anti-Bacterial Agents Cephalosporins Enzyme Models Chemical chemistry Mutation Penicillin Amidase Dimerization Protein Binding |
| Zdroj: | Protein Science |
| DOI: | 10.1110/ps.27502/full |
| Popis: | Glutarylamidase is an important enzyme employed in the commercial production of 7-aminocephalosporanic acid, a starting compound in the synthesis of cephalosporin antibiotics. 7-aminocephalosporanic acid is obtained from cephalosporin C, a natural antibiotic, either chemically or by a two-step enzymatic process utilizing the enzymes D-amino acid oxidase and glutarylamidase. We have investigated possibilities for redesigning glutarylamidase for the production of 7-aminocephalosporanic acid from cephalosporin C in a single enzymatic step. These studies are based on the structures of glutarylamidase, which we have solved with bound phosphate and ethylene glycol to 2.5 A resolution and with bound glycerol to 2.4 A. The phosphate binds near the catalytic serine in a way that mimics the hemiacetal that develops during catalysis, while the glycerol occupies the side-chain binding pocket. Our structures show that the enzyme is not only structurally similar to penicillin G acylase but also employs essentially the same mechanism in which the alpha-amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the O(gamma) through a hydrogen bond relay network involving the alpha-amino group of the serine and a bound water molecule. A plausible model of the enzyme-substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7-aminocephalosporanic acid. |
| Databáze: | OpenAIRE |
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